Lab 1 and 2

Question 1

Why do cells need to regulate their genes?

Answer 1

• external environmental conditions
• developmental cues
• in response to hormone signals

Question 2

What type of gene regulation was investigated in Labs 1 and 2?

Answer 2

transcriptional regulation (the most common type of gene regulation in bacteria)

Question 3

When does transcriptional regulation occur?

Answer 3

in the first stage of gene expression - before significant amounts of mRNA are synthesized

Question 4

What kinds of genes does the ara operon contain?

Answer 4

genes that encode proteins encode proteins required to metabolize a sugar (L-arabinose, which is a pentose sugar)

Question 5

What happens to the genes in the ara operon when L-arabinose is present?

Answer 5

the genes in the ara operon may be transcribed at high levels

Question 6

What happens to the genes in the ara operon when glucose is present?

Answer 6

when a more easily metabolized sugar such as glucose is present, there is little transcription

Question 7

What is the araC gene responsible for in pGLO?

Answer 7

encodes the regulator protein AraC

Question 8

What is the ori responsible for in pGLO?

Answer 8

the origin of replication for the plasmid

Question 9

What is the bla gene responsible for in pGLO?

Answer 9

codes for a protein that makes the bacterial cell resistant to the antibiotic ampicillin

Question 10

What is the GFP gene responsible for in pGLO?

Answer 10

codes for a green fluorescent protein

Question 11

What is the Pbad responsible for in pGLO?

Answer 11

it’s the ara operon promoter

Question 12

What does Pbad do under normal conditions?

Answer 12

regulates the transcription of genes in the ara operon (responsible for the metabolism of arabinose), but in pGLO these regulatory genes have been replaced by the GFP gene

Question 13

How is GFP controlled in this lab?

Answer 13

by the bacterial promoter Pbad

Question 14

What is AraC

Answer 14

an allosteric regulatory protein with two binding sites

Question 15

What happens when only arabinose is present in the environment?

Answer 15

• AraCi (inactive) binds to the sugar
• binding causes a conformational change of AraCi to AraCa (active)
• this permits recognition and binding to aral (activator sequence)

Question 16

What does AraCa do?

Answer 16

facilitates RNA polymerase binding to the Pbad promoter

Question 17

What happens when no arabinose is present in the environment?

Answer 17

1. AraC is not active (stays as AraCi) and cannot bind to the aral sequence
2. RNA polymerase will not bind efficiently to the Pbad sequence
3. very little transcription will occur (little GFP)

Question 18

In the absence of arabinose, what does AraCi do?

Answer 18

functions as a repressor by binding to regions of the operon causing the DNA to form a loop, which prevents the binding of RNA polymerase

Question 19

How do we get the most efficient binding of RNA polymerase to Pbad?

Answer 19

1. two molecules of AraCa must bind to the aral sequence
2. CAP must bind to the cAMP-CAP binding site (CBS)

Question 20

What are the two ways CAP can be found?

Answer 20

1. when cAMP is bound to CAP, CAP is active and can bind to the CBS
2. when cAMP is not bound to CAP, CAP is inactive and cannot bind to CBS

Question 21

What happens when there are high concentrations of glucose in bacterial cells?

Answer 21

it will reduce the synthesis of cAMP and thus CAP is less likely to become activated and bind to the CBS sequence

Question 22

What happens if arabinose is present in high glucose concentrations?

Answer 22

high concentrations of glucose will prevent the formation of CAPa and the binding of RNA polymerase to Pbad
- little transcription of GFP will occur

Question 23

What happens when arabinose is present with low concentrations of glucose?

Answer 23

• two molecules of AraCa will bind to the aral sequence
• CAPa will bind to the CBS sequence
• the binding of RNA polymerase will be efficient
• transcription of GFP will be relatively high

Question 24

How is catabolite repression demonstrated?

Answer 24

CAP ensures that other carbon utilization pathways are not expressed in the presence of glucose because glucose is the preferred carbon source

Question 25

How does the pGLO plasmid get into the bacterial cell?

Answer 25

genetic transformation
- the uptake and incorporation of foreign DNA into the call

Question 26

What are plasmids?

Answer 26

naturally occurring circles of DNA found in bacteria and some fungi and can be transferred between cells, serving as a vector

Question 27

How are plasmids replicated?

Answer 27

by the bacterial cell’s own DNA replication machinery

Question 28

How can we differentiate between bacterial cells containing our plasmid of interest and those that do not?

Answer 28

bla gene
- encodes a bacterial enzyme that renders the bacterial cell resistant to the antibiotic ampicillin

Question 29

What is a Western blot?

Answer 29

A Western blot is a laboratory technique used to detect specific proteins in a sample. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and detecting them using antibodies.

Question 30

What steps are involved in a Western blot procedure?

Answer 30

1. Gel Electrophoresis: Separate proteins based on size.
2. Transfer: Blot proteins onto a membrane (nitrocellulose or PVDF).
3. Blocking: Prevent nonspecific binding by incubating with a blocking solution.
4. Probing: Use primary antibodies to bind to target proteins.
5. Detection: Use secondary antibodies conjugated with enzymes or fluorophores to visualize the proteins.

Question 31

What is the primary purpose of chromatin immunoprecipitation (ChIP)?

Answer 31

ChIP is used to identify and quantify specific protein-DNA interactions, allowing researchers to study transcription regulation through histone modification and transcription factor binding.

Question 32

What is the primary purpose of the Electrophoretic Mobility Shift Assay (EMSA)?

Answer 32

used to study protein-DNA interactions, specifically to identify sequence-specific DNA-binding proteins and to analyze the binding dynamics of these interactions.

Question 33

How does EMSA differentiate between free DNA and protein-DNA complexes?

Answer 33

Protein-DNA complexes migrate more slowly through a gel than free linear DNA fragments due to their larger size and altered conformation, resulting in a “shift” in their mobility.

Question 34

What are the three key steps involved in the EMSA process?

Answer 34

The three key steps are: (1) binding reactions, (2) electrophoresis, and (3) probe detection.